Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)⁺, but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application.     

Redesigning Escherichia coli metabolism for anaerobic production of isobutanol

Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an Escherichia coli strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned E. coli strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing E. coli strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.     

Rational design of a synthetic Entner–Doudoroff pathway for enhancing glucose transformation to isobutanol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner–Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45 g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67 g/L, 0.50 C-mol/C-mol and 0.456 g/(L × h) in a shorter time of 30 h, compared with that of the starting strain E. coli LA09.     

Improving isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with over-expressing NADPH-specific glucose-6-phosphate dehydrogenase (Zwf1)

Isobutanol is a more promising biofuel than ethanol due to its higher energy density and lower hygroscopicity. Saccharomyces cerevisiae, as a model eukaryote, has the potential advantage to produce isobutanol because of its greater tolerance to higher alcohols. NADPH is a key cofactor for isobutanol synthesis, and glucose-6-phosphate dehydrogenase (Zwf1) is one of the main NADPH-supplying sources in S. cerevisiae. In this study, we investigated the effects of over-expressing ZWF1 on isobutanol titers. Our results showed that engineered strain HZAL-7023 produced 6.22 mg isobutanol per g glucose, which increased by 6.64-fold compared with the parent strain, while engineered strain HZAL-7023 22-ZWF1 produced 11.46 mg isobutanol per g glucose, which increased by 1.82-fold compared with engineered strain HZAL-7023. These results suggested that improvement of NADPH supply through over-expressing ZWF1 contributed to isobutanol biosynthesis in S. cerevisiae. These results also verified the proposed concept of increasing isobutanol titers in S. cerevisiae by resolving cofactor imbalance. Finally, this study provides a new strategy for enhancing isobutanol biosynthesis.     

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.     

Production of D-arabitol by a metabolic engineered strain of Bacillus subtilis

A novel method for D-arabitol production with a metabolically engineered Bacillus subtilis strain is described. A known transketolase-deficient and D-ribose-producing mutant of B. subtilis (ATCC 31094) was further modified by disruption of its rpi (D-ribose phosphate isomerase) gene to create a D-ribulose- and D-xylulose-producing B. subtilis strain. Expression of the D-arabitol phosphate dehydrogenase gene of Enterococcus avium in the D-ribulose- and D-xylulose-producing strain resulted in a strain of B. subtilis capable of converting D-glucose to D-arabitol with a high yield (38%) and little by-product formation.     

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180?mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270?mg/L isobutanol and 40?mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14?g/L branched-chain alcohols over the duration of 50?days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.     

Isobutanol production in Corynebacterium glutamicum: Suppressed succinate by-production by pckA inactivation and enhanced productivity via the Entner–Doudoroff pathway

On the basis of our previous studies of microbial L-valine production under oxygen deprivation, we developed isobutanol-producing Corynebacterium glutamicum strains. The artificial isobutanol synthesis pathway was composed of the first three steps of the L-valine synthesis pathway; and the subsequent Ehrlich Pathway: pyruvate was converted to 2-ketoisovalerate in the former reactions; and the 2-keto acid was decarboxylated into isobutyraldehyde, and subsequently reduced into isobutanol in the latter reactions. Although there exists redox cofactor imbalance in the overall reactions, i.e., NADH is generated via glycolysis whereas NADPH is required to synthesize isobutanol, it was resolved by taking advantage of the NAD-preferring mutant acetohydroxy acid isomeroreductase encoded by ilvC^TM and the NAD-specific alcohol dehydrogenase encoded by adhA. Each enzyme activity to synthesize isobutanol was finely tuned by using two kinds of lac promoter derivatives. Efficient suppression of succinate by-production and improvement of isobutanol yield resulted from inactivation of pckA, which encodes phosphoenolpyruvate carboxykinase, whereas glucose consumption and isobutanol production rates decreased because of the elevated intracellular NADH/NAD⁺ ratio. On the other hand, introduction of the exogenous Entner–Doudoroff pathway effectively enhanced glucose consumption and productivity. Overexpression of phosphoenolpyruvate:carbohydrate phosphotransferase system specific to glucose and deletion of ilvE, which encodes branched-chain amino acid transaminase, further suppressed by-products and improved isobutanol productivity. Finally, the produced isobutanol concentration reached 280 mM at a yield of 84% (mol/mol glucose) in 24 h.     

Improving isobutanol production in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> by co-producing ethanol and modulation of pentose phosphate pathway

Redox imbalance has been regarded as the key limitation for anaerobic isobutanol production in metabolically engineered Escherichia coli strains. In this work, the ethanol synthetic pathway was recruited to solve the NADH redundant problem while the pentose phosphate pathway was modulated to solve the NADPH deficient problem for anaerobic isobutanol production. Recruiting the ethanol synthetic pathway in strain AS108 decreased isobutanol yield from 0.66 to 0.29 mol/mol glucose. It was found that there was a negative correlation between aldehyde/alcohol dehydrogenase (AdhE) activity and isobutanol production. Decreasing AdhE activity increased isobutanol yield from 0.29 to 0.6 mol/mol. On the other hand, modulation of the glucose 6-phosphate dehydrogenase gene of the pentose phosphate pathway increased isobutanol yield from 0.29 to 0.41 mol/mol. Combination of these two strategies had a synergistic effect on improving isobutanol production. Isobutanol titer and yield of the best strain ZL021 were 53 mM and 0.74 mol/mol, which were 51 % and 12 % higher than the starting strain AS108, respectively. The total alcohol yield of strain ZL021 was 0.81 mol/mol, which was 23 % higher than strain AS108.     

基元模式分析在生物网络和途径分析中的应用

基元模式分析是应用最广泛的代谢途径分析方法。基元模式分析的研究对象从代谢网络发展到信号传导网络;研究尺度从细胞到生物反应器,甚至生态系统;数学描述从稳态分解到动态解析;研究领域从微生物代谢到人类疾病。以下综述了基元模式分析的算法和软件开发现状,以及其在代谢途径与鲁棒性、代谢通量分解、稳态代谢通量分析、动态模型与生物过程模拟、网络结构与调控、菌株设计和信号传导网络等方面的应用。开发新的算法解决组合爆炸问题,探索基元模式与代谢调控的关系以及提高菌株设计算法效率是今后基元模式的重要发展方向。     

Application of hybrid cybernetic model in simulating myeloma cell culture co-consuming glucose and glutamine with mixed consumption patterns

A dynamic model called hybrid cybernetic model (HCM) based on structured metabolic network is established for simulating mammalian cell metabolism featured with partially substitutable and partially complementary consumption patterns of two substrates, glucose and glutamine. Benefiting from the application of elementary mode analysis (EMA), the complicated metabolic network is decomposed into elementary modes (EMs) facilitating the employment of the hybrid cybernetic framework to investigate the external and internal flux distribution and the regulation mechanism among them. According to different substrate combination, two groups of EMs are obtained, i.e., EMs associated with glucose uptake and simultaneous uptake of glucose and glutamine. Uptake fluxes through various EMs are coupled together via cybernetic variables to maximize substrate uptake. External fluxes and internal fluxes could be calculated and estimated respectively, by the combination of the stoichiometrics of metabolic networks and fluxes through regulated EMs. The model performance is well validated via three sets of experimental data. Through parameter identification of limited number of experimental data, other external metabolites are precisely predicted. The obtained kinetic parameters of three experimental cultures have similar values, which indicates the robustness of the model. Furthermore, the prediction performance of the model is successfully validated based on identified parameters.     

Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.     

Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum

Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol)(-1), whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol)(-1). Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol)(-1). A further increase is possible by the application of additional C1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C1 carbon and sulfur (0.91 (C-mol) (C-mol)(-1)). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g(-1) h(-1), as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g(-1) h(-1). The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.     

应用最大熵原理通过酶控制通量预测突变体的通量分布

在代谢工程和系统生物学领域, 计算机模拟比以往更为有效的应用于生物过程的分析和优化。胞内代谢通量可以用代谢通量分析和基元模式分析来估算。由于测定数据的不足和误差, 以及基元途径的冗余, 经常很难得到准确的代谢通量分布数据。本研究提出一种基于最大熵原理的算法来计算基元模式系数。欠定和不确定条件下, 通过胞外代谢通量数据估算胞内代谢通量分布。为了检验算法的可行性, 对杂交瘤细胞、枯草芽孢杆菌和大肠杆菌的胞内代谢通量分布做了估算。本研究提出的基于最大熵原理的优化算法避免了对细胞状态的生理学假设。与其他目标函数相比, 可以更为可靠和可行的估算胞内代谢通量分布。     

Metabolic network properties help assign weights to elementary modes to understand physiological flux distributions

MOTIVATION: Elementary modes (EMs) analysis has been well established. The existing methodologies for assigning weights to EMs cannot be directly applied for large-scale metabolic networks, since the tremendous number of modes would make the computation a time-consuming or even an impossible mission. Therefore, developing more efficient methods to deal with large set of EMs is urgent. RESULT: We develop a method to evaluate the performance of employing a subset of the elementary modes to reconstruct a real flux distribution by using the relative error between the real flux vector and the reconstructed one as an indicator. We have found a power function relationship between the decrease of relative error and the increase of the number of the selecting EMs, and a logarithmic relationship between the increases of the number of non-zero weighted EMs and that of the number of the selecting EMs. Our discoveries show that it is possible to reconstruct a given flux distribution by a selected subset of EMs from a large metabolic network and furthermore, they help us identify the 'governing modes' to represent the cellular metabolism for such a condition.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942

Glycogen synthesis initiated by glucose-1-phosphate adenylyltransferase (glgC) represents a major carbon storage route in cyanobacteria which could divert a significant portion of assimilated carbon. Significant growth retardation in cyanobacteria with glgC knocked out (ΔglgC) has been reported in high light conditions. Here, we knocked out the glgC gene and analyzed its effects on carbon distribution in an isobutanol-producing strain of Synechococcus elongatus PCC7942 and its parental wild-type strain. We showed that isobutanol production was able to partially rescue the growth of ΔglgC mutant where the growth rescue effect positively correlated with the rate of isobutanol production. Using NaH¹⁴CO₃ incorporation analysis, we observed a 28 % loss of total carbon fixation rate in the ΔglgC mutant compared to the wild-type. Upon expression of the isobutanol production pathway in ΔglgC mutant, the total carbon fixation rate was restored to the wild-type level. Furthermore, we showed that 52 % of the total carbon fixed was redirected into isobutanol biosynthesis in the ΔglgC mutant expressing enzymes for isobutanol production, which is 2.5 times higher than that of the wild-type expressing the same enzymes. These results suggest that biosynthesis of non-native product such as isobutanol can serve as a metabolic sink for replacing glycogen to rescue growth and restore carbon fixation rate. The rescue effect may further serve as a platform for cyanobacteria energy and carbon metabolism study.     

Metabolic flux analysis of Escherichia coli expressing the Bacillus subtilis acetolactate synthase in batch and continuous cultures.

Metabolically engineered Escherichia coli expressing the B. subtilis acetolactate synthase has shown to be capable of reducing acetate accumulation. This reduction subsequently led to a significant enhancement in recombinant protein production. The main focus of this study is to systematically examine the effect of ALS in the metabolic patterns of E. coli in batch and continuous culture. The specific acetate production rate of a strain carrying the B. subtilis als gene is 75% lower than that of the control strain (host carrying the control plasmid pACYC184) in batch cultures. The ALS strain is further demonstrated to be capable of maintaining a reduced specific acetate production rate in continuous cultures at dilution rates ranging from 0.1 to 0.4 h-1. In addition, this ALS strain is shown to have a higher ATP yield and lower maintenance coefficient. The metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin.